I confirm the necessity to run the gel with a specific ladder for RNA, but it is necessary go ahead to verify that is real RNA and good enough for real-time or other analysis. The most important tests are

1. synthesis of cDNA

2. PCR

If he will not get good results from these two tests, what he got is not RNA of good quality, even if the gel could say the contrary.

I suppose, that you Arthur, agree with me.

Bear in mind that 80-95% of total RNA is ribosomes, so you're seeing two sharp bands because those two RNA species make up the vast, overwhelming majority of the RNA present, and they're both of fixed size. mRNA is much lower abundance, and the size of mRNAs varies enormously, so even when you DO see a faint background smear, it is usually very very faint, very backgroundy, and very smeared.

I would never expect to assess my mRNA content by gel electrophoresis: this gel simply says "yep, you have RNA, and it's not degraded." If you don't see ribosomal bands, you definitely have problems, but that's about the most information you could expect to get from this.

It thus strongly implies that you have mRNA, but isn't a priori proof.

Also, I'm assuming you mean cDNA synthesis rather than in vitro transcription? Assuming your 260/230 ratio is good, I'd go right ahead. 

The gel image appears good .you resolve in longer time and see any smear is appearing. as per my knowledge you can not see mRNA on the gel since it is  small in size.

all the best 

What I see from the gel is that the RNA is not degradated. You assume that there is good mRNA on the base of good quality of more abundant rRNA. It is very difficult see mRNA on the gel, is still difficult see it on Bioanalyzer microfluidic electrophoresis.

Your RNA looks very good to be processed. Procede with cDNA syntesis using oligodT, and on the base of cDNA you get you will know that in your sample there is also mRNA. Don't worry, the pattern you got is the pattern scientists  get normally.

Look at this pubblication: A Simple and Effective Method for High Quality Co-

Extraction of Genomic DNA and Total RNA from Low

Biomass Ectocarpus siliculosus, the Model Brown Alga

Maria Greco

Thank you all for your kind support and advice.

The ladder could confirm that the two bands are of the same size of typical Total RNA extracted from a tissue, but what is more important is the pattern of bands of total RNA extracted, and this is perfect and the integrity that is also perfect.

If the two bands you see are not RNA what they are? This is not a product of PCR is a nucleic acid extraction. Therefore, DNA and RNA. We know that are not DNA, so, the two bands we see are for sure total RNA.

But just to confirm what is obvious, you can run the gel with a ladder. But more important do the cDNA of one sample and perform a PCR to verify if it is retrotranscribed and of good quality.

I confirm the necessity to run the gel with a specific ladder for RNA, but it is necessary go ahead to verify that is real RNA and good enough for real-time or other analysis. The most important tests are

1. synthesis of cDNA

2. PCR

If he will not get good results from these two tests, what he got is not RNA of good quality, even if the gel could say the contrary.

I suppose, that you Arthur, agree with me.

Dear Zainab,

In this photo I see two fractions of rRNA, 28s on the top, 18s on the bottom. mRNA is not visible.

Bests,

Hovsep

Hi Zeinab,

May I have your protocol for non denaturing gel elechtrophoresis! I extracted RNA and need to run it on the gel before RTpcr. I have tried 1% agaros gel in order to determine RNA integrity, but I just saw one band! I should also mention that I used 5x DNA loading buffer! However I think I should see 18 and 28S in two different lines! likewise I did not denature RNA and applied on the gel without any special treatment!

Your gel seems clear. Could you please let me know your protocol!

Elar: I would caution against replying to 3 year old posts, but since you asked: RNA can be run successfully under normal (non-denaturing) conditions provided you denature it first.

Prepare a 1% agarose gel as normal (for DNA, etc).

Prepare a loading mix with 1-2ul of your RNA, 2ul of 5x DNA loading buffer, and 6-7ul of formamide.

(formamide is a denaturant, so you want to aim for 60-70% final)

Heat this to 65 degrees for 5 mins (this denatures secondary structure: 65 degrees is the hottest you can really get RNA before it begins to degrade).

Remove from heat block, place IMMEDIATELY on ice. This prevents secondary structures reforming.

Load these samples on your gel, run it.

Thank you so much John.